A combination of serum leucine-rich α -2-glycoprotein 1, CA19-9 and interleukin-6 differentiate biliary tract cancer from benign biliary strictures

Cholangiocarcinoma (CCA) and gallbladder carcinoma are often grouped together as biliary tract cancer (BTC) (de Groen et al, 1999). CCA is a rare but devastating disease that accounts for ∼15% of primary liver malignancies (Shaib and El-Serag, 2004). Although the incidence in Western nations has been estimated at 1 per 100 000, there are as yet unexplained increases in the age-adjusted incidence and mortality rates of intrahepatic CCA (Khan et al, 2002b; Shaib et al, 2004). Disorders of the bile ducts associated with inflammation, stricturing and cholestasis, such as primary sclerosing cholangitis (PSC), have been identified as risk factors for BTC (Broome et al, 1995; Kornfeld et al, 1997; Bergquist et al, 2002; Burak et al, 2004; Claessen et al, 2009). Other inflammatory biliary diseases such as immunoglobulin G4-associated cholangitis (IAC) are also difficult to distinguish from BTC and PSC, occasionally leading to incorrect diagnosis and management. At present, surgical resection is the only curative treatment for BTC, with 5-year survival rates after R0 resection for hilar CCA of 11–41% and for distal extrahepatic CCA of 27–37% (Nakeeb et al, 1996; Neuhaus et al, 1999; Jarnagin et al, 2001; Nagorney and Kendrick, 2006; DeOliveira et al, 2007). Unresectable disease has an associated 5-year survival of less than 10% (Neuhaus et al, 1999; Reddy and Patel, 2006), and confirms the need for more accurate diagnostic biomarkers. The gold standard for the diagnosis of BTC is cytological or histological confirmation of malignancy within the biliary stricture, either via endoscopic retrograde cholangiopancreatography (ERCP) or percutaneously (Khan et al, 2002a). However, this can be difficult to achieve due to the complex anatomical relationship between the liver hilum and bile ducts and the mode of tumour extension, which is often not mass forming. Approximately 15% of resections performed for suspected, pathologically unconfirmed, hilar BTC are due to benign strictures (Gerhards et al, 2001; Reddy and Patel, 2006). Carbohydrate antigen 19-9 (CA19-9) is an established serum marker for the diagnosis of BTC, although it is reported to have a wide variation in sensitivity (50–90%) and specificity (54–98%), (Chalasani et al, 2000; Patel et al, 2000; Levy et al, 2005) and is often falsely elevated in benign biliary disease and/or cholangitis, with levels falling after relief of biliary obstruction and sepsis. Additionally, CA19-9 is virtually undetectable in the 7% of the population who are negative for the Lewis antigen (Steinberg, 1990). There is consequently a need to identify better circulating biomarkers for this disease. However, tumour marker proteins may be present at low concentrations in the circulation and masked by abundant blood proteins in proteomic analyses. Consequently, researchers have coupled immunoaffinity depletion with proteomic profiling to specifically remove abundant proteins and increase proteomic coverage (Liu et al, 2006; Qian et al, 2008). This study analysed differential protein expression in four different pools of serum from patients with BTC, PSC, IAC and healthy volunteers. Using proteomic analysis the putative marker leucine-rich α-2-glycoprotein (LRG1) was shown to be upregulated in BTC, and was validated using a serum enzyme-linked immunosorbent assay (ELISA). The ability of LRG1 to differentiate BTC from benign biliary disease was evaluated and compared with the current standard tumour marker CA19-9, while taking into consideration potentially confounding conditions such as biliary obstruction and inflammation. Immunohistochemistry for LRG1 was performed on normal hepatobiliary tissue, benign biliary disease (PSC and primary biliary cirrhosis (PBC)) and BTC to evaluate differential staining characteristics. Serum interleukin-6 (IL-6) was also measured as it has been recently shown to be a key inflammatory cytokine involved in the carcinogenesis of CCA and may induce LRG1 (Hodge et al, 2005; Shirai et al, 2009).